In previous studies, we provided evidence that the cytoskeleton provides an important organizational component of receptor-mediated endocytosis in hepatocytes. In particular, receptor-mediated endocytosis of asialoorosomucoid (ASOR) by hepatocytes results in formation of early endocytic vesicles that undergo fission, with one daughter vesicle that contains most of the receptor recycling back to the cell surface, while the other daughter vesicle that contains most of the ligand, trafficking to the lysosome where ligand is degraded. During the past funding period, we showed that endocytic vesicles bind to and move along microtubules. We (reconstituted this process as well as the process of early endocytic vesicle fission and segregation in vitro using a novel fluorescence microscopy system that we developed. These studies showed that kinesins, both plus and minus-end directed, provided the force for this process. We now propose to extend these studies to dissect mechanistically those cellular components that are required for vesicle processing. This will include studies in which these events have been reconstituted from well-defined subcellular constituents. We will utilize similar technology to continue our mechanistic studies of Herpes virus egress. In the previous funding period we developed assays to monitor both the assembly of enveloped HSV particles and their traffic through the hepatocyte cytoplasm. We also established techniques to enable the isolation of HSV-containing cytoplasmic organelles from infected HuH-7 cells. These techniques should enable us to extend these studies to the Trfl and Trfl trafficking mutants. The Specific Aims of this application are: (1) to characterize endosome/microtubule interaction and motility at specific stages of the endocytic process and to determine and quantify direction of motility, motors, and accessory regulatory proteins; (2) to reconstitute endocytic vesicle motility and fission from defined components in vitro and to determine the regulatory role of phosphorylation in these processes; and (3) to examine the cellular components that are required for trafficking of Herpes simplex virus (HSV) from the nucleus to the cell surface.